Abstract
In radiation biophysics, it is a subject of nowadays research to investigate DNA strand break repair in detail after damage induction by ionizing radiation. It is a subject of debate as to what makes up the cell’s decision to use a certain repair pathway and how the repair machinery recruited in repair foci is spatially and temporarily organized. Single-molecule localization microscopy (SMLM) allows super-resolution analysis by precise localization of single fluorescent molecule tags, resulting in nuclear structure analysis with a spatial resolution in the 10 nm regime. Here, we used SMLM to study MRE11 foci. MRE11 is one of three proteins involved in the MRN-complex (MRE11-RAD50-NBS1 complex), a prominent DNA strand resection and broken end bridging component involved in homologous recombination repair (HRR) and alternative non-homologous end joining (a-NHEJ). We analyzed the spatial arrangements of antibody-labelled MRE11 proteins in the nuclei of a breast cancer and a skin fibroblast cell line along a time-course of repair (up to 48 h) after irradiation with a dose of 2 Gy. Different kinetics for cluster formation and relaxation were determined. Changes in the internal nano-scaled structure of the clusters were quantified and compared between the two cell types. The results indicate a cell type-dependent DNA damage response concerning MRE11 recruitment and cluster formation. The MRE11 data were compared to H2AX phosphorylation detected by γH2AX molecule distribution. These data suggested modulations of MRE11 signal frequencies that were not directly correlated to DNA damage induction. The application of SMLM in radiation biophysics offers new possibilities to investigate spatial foci organization after DNA damaging and during subsequent repair.
Highlights
When in the early 1930s Ernst Ruska and Max Knoll developed the first electron microscope [1], super-resolution started its triumph into biological and biomedical research
MRE11 foci were analyzed to obtain further insights into the assembling of the proteins which undergo multiple functional processes accompanied by homo-dimerization under certain conditions
After radiation-induced DNA damaging, MRE11 as a member of the MRN complex is primarily involved in Homologous recombination repair (HRR) and alternative non-homologous end joining (a-Non-homologous end joining (NHEJ)) of DNA double-strand breaks [51], but the MRN complex is a prominent actor in radiation response
Summary
When in the early 1930s Ernst Ruska and Max Knoll developed the first electron microscope [1], super-resolution started its triumph into biological and biomedical research. With the development of nowadays fluorescent dyes and multiple fluorescent proteins, it has become possible to separate single fluorescent tags of specific single-molecule labels. The potentials of the combination of instrumentation, specific specimen labelling strategies and appropriate fluorescent tags induced the developments of a variety of novel techniques such as, for instance PALM Since the preparation conditions of specimens for most SMLM techniques are very similar to those applied in standard fluorescence microscopy, SMLM offers broad perspectives of applications in many fields of biological and biomedical research as well as in diagnostics [13,14]
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